Rev Argent Microbiol, 1983, 15(2), 65 - 71 {Acetobutylic fermentation: strains and regional raw materials}; Benassi FO et al.; The purpose of the present work was to show, as a first stage, that it is possible to characterize autochtohnous strains of Clostridium acetobutilicum of a good solvent producing capacity, specially N-butanol, through the utilization of suitable techniques for isolating anaerobic microorganisms . Cassava roots were employed as raw material using suitable culture media and an anaerobic jar of cold catalyst . The fermentative capacity of the strains thus isolated was evaluated against a control strain of Clostridium acetobutilicum . Even though some of the strains showed a greater solvent producing power, most of them showed lower fermentation capacity than the control strain, which could be increased, by applying successive thermic treatments . As a second stage, and due to the low cost production of cassava in the Province of Misiones, we studied its utilization as an acetone-butanol fermentation substrate . Mashes composed of binary mixtures of cassava flour and variable amounts of integral flour maize or soy were treated with selected "starters" of Clostridium acetobutilicum, being further processed according to standardized techniques in order to obtain the already mentioned solvents . Mashes concentration influence was also studied using culture media the composition of which proved to be excellent in all experiments carried out under "static system" conditions . The highest fermentative yields (maximum value recorded: 26,20 g of total solvents, with respect to dry solids), were recorded for mashes obtained from mixtures containing integral maize flour; these showed a higher degree of nutrients utilization than those prepared with integral soy flour. Rev Argent Microbiol, 1983, 15(1), 51 - 5 {Identification of strain B 657 of Clostridium botulinum}; Gimenez DF et al.; Strain 657 has been described as a toxin variant of Clostridium botulinum type B . Toxin neutralization tests performed with types B and A botulinum antitoxins of known potency, avidity and specificity at 20, 200, 2.000 and 20.000 DL50/mouse level of testing have shown that 657 toxin is a mixture of B (90 to 99% of the complex) and A antigenic fractions . The possibility of a cross contamination between A and B serotypes has been, in principle, ruled out through the serologic screening of 33 toxin samples coming one each from 33 well isolated colonies taken from two colony variants of this strain . Strain 657 produces a new complex toxin and is proposed as the candidate for the prototype of a new C . botulinum serotype: subtype Ba. Z Erkr Atmungsorgane, 1983, 161(1), 28 - 35 {The contribution of Robert Koch to the founding of the doctrine of disinfection and present priorities in disinfection research and practice}; Weuffen W; Robert Kochs studies on disinfection are distinguished by well defined, clear experimental arrangement and commitment to practice . He investigated the effectiveness of disinfection by following recultivation tests in vitro and in animal experiments . He examined chemical substances of different origin, the action of hot air and superheated steam with a spectrum of microorganisms, including bacterial spores . Priorities of the recent development in disinfection are among other things the application of steam disinfectors and of chemical disinfectants in the gas-phase e.g . for disinfection of tubes, the use of combinations of tensides with disinfectants, of disinfectants effective against spores of clostridium and against hepatitis viruses, of disinfectants with low corrosiveness and low toxicity against men and domestic animals . Disinfection is a component of a system of measures in antimicrobial regimes and in protective systems. Toxicon, 1983, 21(4), 566 - 9 Amino acid composition of Clostridium botulinum type F neurotoxin; DasGupta BE et al.; To develop reliable data on the amino acid composition of type F botulinum neurotoxin, three batches of the neurotoxin were analyzed . Each batch was isolated from a separate neurotoxin producing bacterial culture . Two batches had inoculum from one source and the other batch one from a different source . Two batches of the neurotoxin were purified by the same method and one was purified by a different method . The neurotoxin preparations were found comparable in purity and similar in amino acid composition . The best estimate of number of amino acid residues per neurotoxin molecule (mol . wt . 155,000) was: Asp218 Thr80 Ser105 Glu128 Pro47 Gly69 Ala47 Val72 CyS9 Met14 Ile128 Leu104 Tyr86 Phe60 Lys90 His13 Arg51 Trp23. Scand J Gastroenterol Suppl, 1983, 85, 37 - 47 Anaerobic bacteria as cause of infections in female genital organs; Bergan T; Anaerobic bacteria constitute a substantial component of the normal vaginal flora and of the outer cervical canal . Consequently, one would expect infections emanating from the vaginal flora to be caused to a substantial degree by anaerobic bacteria . The anaerobes may contribute in colpitis, but their role is difficult to prove in this situation, since sampling only yields normal flora components . One clue that anaerobes may contribute to colpitis is the circumstance that the flora under those conditions differ from the normal situations . Another is the circumstance that metronidazole, which only inhibits anaerobes, does reduce symptoms e.g . of colpitis in the presence of the microaerophilic Gardnerella vaginalis, which is resistant to metronidazole (but sensitive to the metabolite hydroxymetronidazole) . In the US, pelvic inflammatory disease (PID) is to a substantial degree apparently caused by anaerobic bacteria . Our experience in Scandinavia indicates that anaerobes are of neglible importance in this condition, since we rarely isolate anaerobes in spite of adequate sampling, transport and processing of specimens . We mostly find anaerobes in post-operative PID when the vaginal wall has been passed, or as a complication of pregnancy (puerperal fever, early rupture of membranes, abortion), and in connection with intrauterine devices . The role of anaerobes in bartholinitis is established . PID may be associated with appendicitis or colonic diverticulosis . Anaerobes are regularly isolated from abscesses developing in the pelvic organs . This also applies to tuboovarial and vulvovaginal abscesses . The most important anaerobic bacteria in infections of the female genital organs are Bacteroides fragilis and other species of the fragilis group, B . melaninogenicus and related species, other Bacteroides, peptococci and peptostreptococci . Clostridium perfringens rarely cause infections of female genital organs, although such conditions have a dramatic course. J Clin Microbiol, 1983 Jan, 17(1), 13 - 5 Quantities of Clostridium botulinum organisms and toxin in feces and presence of Clostridium botulinum toxin in the serum of an infant with botulism; Paton JC et al.; A 7-week-old boy presented with symptoms and signs characteristic of infant botulism, and the diagnosis was confirmed by the detection of Clostridium botulinum type A organisms and toxin in the feces . The levels of organisms and toxin in the feces were measured throughout the 81-day period in hospital . The maximum levels detected were 2.46 x 10(8) C . botulinum type A colony-forming units and 64,000 mouse 100% lethal doses of type A toxin per g (wet weight) of feces . C . botulinum toxin was also detected in two samples of the patient's serum, collected 3 and 10 days after admission . Improvement in the patient's clinical condition occurred before the levels of organisms and toxin in the feces reached their maxima . A slight improvement may also have occurred while toxin was still present in the serum. Ann Otol Rhinol Laryngol, 1983 Jan-Feb, 92(1 Pt 1), 91 - 6 Organic acids and anaerobic microorganisms in the contents of the cholesteatoma sac; Iino Y et al.; Organic acids in the contents of the cholesteatoma sac from 28 cases were studied by gas chromatographic technique . Five volatile fatty acids (acetate, propionate, isobutyrate, butyrate and isovalerate) and lactate were detected in large amounts, which may lower the pH of the cholesteatoma content . These acids were considered to be derived from products of anaerobic microorganisms . Therefore, the contents from 12 cases were cultured anaerobically in a glove box . Obligate microorganisms were identified in 92% of the cases and Peptococcus, Bacteroides, and Clostridium species were frequently isolated . In vitro, such obligate anaerobes produced various organic acids from the cholesteatoma content . Facultatives such as Staphylococcus aureus and Proteus mirabilis produced acetate in the content under aerobic and anaerobic conditions, whereas no organic acid was produced by Pseudomonas aeruginosa . Organic acids in the cholesteatoma content could be fermentative products made by the microorganisms, anaerobes and facultatives, which use the content as a substrate for acid production. Ann Rech Vet, 1983, 14(4), 408 - 11 Diarrhea in one to three week-old piglets associated with Clostridium perfringens type A; Nabuurs MJ et al.; Investigation on the aetiology of diarrhoea in piglets of one to three weeks of age revealed high numbers of Clostridium perfringens type A in intestinal contents of severely affected animals . Experimental infections with hysterectomy derived, colostrum deprived piglets performed in an isolator resulted in a clinical picture indistinguishable from the clinical signs observed under field conditions i.e . creamy diarrhoea, emaciation, abundant gas in the gut but usually low mortality rate . The predominant post-mortem findings were the presence of gas in the intestinal lumen and in the mucosa, superficial necrosis and villus atrophy. Int J Biochem, 1983, 15(11), 1345 - 51 Preparation of neuraminidase-resistant human alpha 1-protease inhibitor and its clearance in rat blood circulation; Omichi K et al.; The sialic acid residues of human alpha 1-protease inhibitor were modified by periodate oxidation and subsequent reductive amination with ethanolamine and sodium cyanoborohydride . The modified inhibitor retained its original trypsin inhibitory activity and was not digested by neuraminidase from Clostridium perfringens . The modified inhibitor disappeared from rat blood circulation at the same rate as the native inhibitor. Gynecol Obstet Invest, 1983, 15(2), 103 - 18 The role of phospholipids in the binding of oxytocin to its receptors in lactating rabbit mammary gland; Warr JL et al.; Phospholipase C (Clostridium welchii and Bacillus cereus) treatment of lactating rabbit mammary gland membranes (140,000 g pellet and sucrose density gradient purified plasma membranes) resulted in a large decrease in the binding of {3H}oxytocin to these subcellular fractions . This decrease was not due to a solubilization of oxytocin receptors but was the result of the removal of phospholipids which may participate in the hormone-receptor interaction . Phospholipase C treatment of the membrane fractions resulted in a dose-dependent removal of different classes of phospholipids . Sphingomyelin, phosphatidylcholine and phosphatidylethanolamine were removed by phospholipase C (C . welchii and B . cereus) treatment . No significant change was observed in the content of phosphatidylinositol . Phospholipase C from B . cereus reduced the content of phosphatidylserine, while the enzyme from C . welchii did not. J Pharmacol Exp Ther, 1983 Jan, 224(1), 135 - 40 Botulinum neurotoxin type E: studies on mechanism of action and on structure-activity relationships; Simpson LL et al.; Single chain type E botulinum neurotoxin was isolated from culture fluids of Clostridium botulinum (strain Alaska E-43) . The neurotoxin, which migrated as a single band in polyacrylamide gel electrophoresis with sodium dodecylsulfate, had a molecular weight of approximately 147,000 . Single chain type E neurotoxin that was exposed to trypsin was converted to a dichain molecule . Pretreatment of the single chain molecule with 1,2-cyclohexanedione, a reagent that selectively modifies arginine residues, inhibited trypsin-induced generation of the dichain molecule . In dose-response experiments (10(-13) to 10(-9) M) on the isolated neuromuscular junction (phrenic nerve-hemidiaphragm preparation), the dichain neurotoxin was approximately two orders of magnitude more potent than the single chain neurotoxin . Neither specie of neurotoxin (1 pmol/mouse, in vivo; 1 X 10(-11) M, in vitro) was very effective in blocking autonomic transmission (vagus nerve-atrium preparation) . The neuromuscular blocking action of the dichain molecule was divided into a sequence of three steps . There was an initial binding step that was relatively rapid, little influenced by temperature and which left the neurotoxin partially accessible to the neutralizing effects of antitoxin . There was a translocation step that was temperature dependent, antagonized by ammonium chloride and methylamine hydrochloride and which caused the neurotoxin to become inaccessible to the neutralizing effects of antitoxin . Finally, there was an intracellular lytic step, during which the toxin blocked excitation-secretion coupling. J Neurochem, 1983 Jan, 40(1), 267 - 75 Attenuation of enkephalin activity in neuroblastoma X glioma NG108-15 hybrid cells by phospholipases; Law PY et al.; The role of membrane phospholipids in enkephalin receptor-mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity in neuroblastoma X glioma NG108-15 hybrids was studied by selective hydrolysis of lipids with phospholipases . When NG108-15 cells were treated with phospholipase C from Clostridium welchii at 37 degrees C, an enzyme concentration--dependent decrease in adenylate cyclase activity was observed . The basal and prostaglandin E1 (PGE1)-stimulated adenylate cyclase activities were more sensitive to phospholipase C (EC 3.1.4.3) treatment than were the NaF-5'-guanylylimidodiphosphate (Gpp(NH)p)-sensitive adenylate cyclase activities . Further, Leu5-enkephalin inhibition of basal or PGE1-stimulated adenylate cyclase activity was attenuated by phospholipase C treatment, characterized by a decrease of enkephalin potency and of maximal inhibitory level . {3H}D-Ala2-Met5-enkephalinamide binding revealed a decrease in receptor affinity with no measurable reduction in number of binding sites after phospholipase C treatment . Although opiate receptor was still under the regulation of guanine nucleotide after phospholipase C treatment, adenylate cyclase activity was more sensitive to the stimulation of Gpp(NH)p . Thus, the reduction of opiate agonist affinity was not due to the uncoupling of opiate receptor from N-component . Further, treatment of NG108-15 hybrid cell membrane with phospholipase C at 24 degrees C produced analogous attenuation of enkephalin potency and efficacy without alteration in receptor binding . The reduction in enkephalin potency could be reversed by treating NG108-15 membrane with phosphatidylcholine, but not with phosphatidylserine, phosphatidylinositol, or cerebroside sulfate . The enkephalin activity in NG108-15 cells was not altered by treating the cells with phospholipase A2 o phospholipase C from Bacillus cereus . Hence, apparently, there was a specific lipid dependency in enkephalin inhibition of adenylate cyclase activity. Arch Immunol Ther Exp (Warsz), 1983, 31(4), 531 - 9 Antigenic hemagglutination inhibition test in examining of the culture supernatants of Clostridium botulinum types A, B, E, and F and some related bacterial species; Reiss J et al.; The hemagglutination inhibition test with using lyophilized preparations of the double botulinal indicator system (antigen sensitized erythrocytes and type-specific homologous antitoxic globulin) was used to the identification of the Cl . botulinum types A, B, E and F culture supernatants and the supernatants of other related bacteria . In the examined indicator hemagglutination system, the type-specific results were obtained in spite of the existence of antigenic cross-reactivity known inside the genus Clostridium . Absence hemagglutination inhibition reaction of the botulinal indicator system with cultured supernatants of the protein A - positive Staph . aureus strains was discussed. Neoplasma, 1983, 30(6), 667 - 80 Allogenic modification of rat sarcoma cells in vitro by staphylococcal exotoxin; Hlavayova E et al.; The effect of toxin preparations from Clostridium welchii and Staphylococcus aureus on the growth of some experimental animal tumor cell lines was investigated . While clostridium toxin exerted considerable cytotoxicity, it did not influence either in vivo or in vitro growth of recovered cells . However, staphylotoxin treated sarcoma cells, while showing normal in vitro growth and metabolism, exhibited decreased growth rates when transplanted into susceptible hosts . This effect was demonstrable even after as many as 10 or more passages in vitro after the toxin treatment . Increased immunogenicity of staphylotoxin treated sarcoma cells was demonstrated by the complement-dependent serum cytotoxicity test . These results indicate that bacterial products, besides cytotoxicity and macrophage stimulation, may exert other effects upon tumor cells, for instance alteration of their immunogenicity and thus influence the tumor growth. J Neurosci Res, 1983, 9(4), 401 - 12 Ganglioside-basic protein interaction: protection of gangliosides against neuraminidase action; Yohe HC et al.; The ability of acidic phospho- and sphingolipids to interact with basic proteins was studied by double diffusion analysis . The phospholipids, tri- and diphosphoinositide, and the sphingolipid, sulfatide, interacted with myelin basic protein as evidenced by precipitin line formation . Of the sialoglycosphingolipids (gangliosides) tested, only the myelin-specific monosialoganglioside, GM4, formed a precipitin line with myelin basic protein . In addition, myelin basic protein retarded the activity of Clostridium perfringens neuraminidase against GM4 and the disialoganglioside, GD1b . Examination of purified rat brain myelin suggested the presence of a neuraminidase activity intrinsic to myelin . This finding, in concert with ganglioside-myelin basic protein complexes which selectively protect against neuraminidase, may provide a physiological explanation for the simplified ganglioside pattern found in myelin. Rev Argent Microbiol, 1983, 15(3), 163 - 8 {Staphylococcal coagglutination for the detection of the enterotoxin of Clostridium perfringens}; Dobosch D; The staphylococcal coagglutination technique has been used in recent years for detecting various bacterial antigens and toxins . It is a simple and rapid technique which requires no specialized equipment . This report describes a staphylococcal coagglutination procedure for assaying C . perfringens enterotoxin . Its sensitivity and specificity were studied with the aid of various enterotoxin preparations and culture filtrates of C . perfringens and other Clostridium species . This assay detects between 1.9 and 3.8 micrograms of enterotoxin per ml . Only the enterotoxin preparations and the C . perfringens culture fluids were positive. Cancer Drug Deliv, 1983, 1(1), 21 - 36 Combination chemotherapy with Clostridium perfringens phospholipase C and cytosine antimetabolites: complementary inhibition directed at membrane lipids; Lee MH et al.; Tumor cell membranes were susceptible to the action of Clostridium perfringens phospholipase C, and this was reflected by inhibition of cellular replication in culture . The differential susceptibility of two neoplastic cell lines to this enzyme was studied in detail . The growth of sarcoma 180 cells cultured in Fischer's medium was markedly inhibited by phospholipase C; whereas, in contrast, cultured L1210 leukemia cells were relatively resistant to the cytotoxic effects of this enzyme . The differential sensitivity of these two neoplastic cell lines to phospholipase C was corroborated by dye-exclusion tests . Thus, leukemia L1210 cells exposed to a concentration of 0.2 mg of phospholipase C per ml of Fischer's medium for 30 min at 37 degrees C were able to exclude Trypan Blue; whereas, only about 21% of sarcoma 180 cells treated under identical conditions were able to exclude the dye . That the cytotoxicity of phospholipase C to sarcoma 180 was the result of hydrolysis of phospholipids of the plasma membrane was supported by measurements of the rate of hydrolysis of radioactivity from the phospholipid of neoplastic cells prelabeled with {3H}choline . Eighty-two percent of incorporated radioactive choline was released from sarcoma 180 cells treated with phospholipase C in Fischer's medium, whereas, only 20% of the label from {3H}choline was solubilized from L1210 leukemia cells treated with the enzyme under similar conditions . Scanning electron microscopy revealed significant damage to sarcoma 180 cells exposed to phospholipase C in Fischer's medium, which was characterized by alterations in size and shape of cells, disappearance of microvilli, and appearance of fistulas in cell membranes; relatively resistant L1210 leukemic cells did not appear to be markedly damaged by comparable enzyme treatment . Exposure of leukemia L1210 cells to phospholipase C in Puck's saline A increased the sensitivity of these cells to enzymatic action . Under these conditions, a comparable amount of phospholipid was hydrolyzed from surface membranes of sarcoma 180 and leukemia L1210 cells, and the degree of membrane damage appeared to be similar, as measured by the capacity of the tumor cell lines to exclude Trypan Blue and by scanning electron microscopy . The extensive damage to membranes by hydrolysis of phospholipids was not accompanied by a change in the degree of specific binding of {3H}concanavalin A(ConA).(ABSTRACT TRUNCATED AT 400 WORDS) Lancet, 1982 Dec 18, 2(8312), 1388 - 91 Peritonitis in continuous ambulatory peritoneal dialysis . Laboratory and clinical studies; Gokal R et al.; During a three year period, 1979-81, 82 patients were treated by continuous ambulatory peritoneal dialysis (CAPD) . The incidence of peritonitis was reduced significantly during the three years from one episode per 20 patient-weeks to one episode every 37 patient-weeks . 83% of the 136 episodes of peritonitis were treated successfully by antibiotic therapy alone . 62% of the total episodes were managed successfully with intraperitoneal cefuroxime . in 13 (16%) patients, CAPD failed because of peritonitis . Hospital admission for peritonitis has been reduced to a mean of 4.3 days per patient per year of CAPD . Fron January to September, 1982, Clostridium-difficile colitis developed in 13 patients . This complication was associated with considerable mortality and morbidity and has prompted a change in antibiotic policy . Patients with peritonitis are now given intraperitoneal netilmicin and intravenous vancomycin . Peritonitis remains the main complication of CAPD, but can be minimised by development of adequate facilities for performing CAPD. Biochim Biophys Acta, 1982 Dec 8, 693(1), 195 - 204 Ethanol-induced changes in the membrane lipid composition of Clostridium thermocellum; Herrero AA et al.; When ethanol is added to the growth medium of Clostridium thermocellum ATCC 27405 and C9, a different membrane composition is observed after the period of growth arrest . Changes in fatty acid composition and some unsaturated, branched hydrocarbons have been monitored by GLC-MS . There is a marked increase in normal and anteiso-branched fatty acids at the expense of isobranched fatty acids and an increase in short and unsaturated fatty acids . Thus, an adaptive response to growth in the presence of ethanol induces a membrane containing fatty acids with lower melting points and produces a more 'fluid' membrane . The suggestion is made that these membrane changes may be maladaptive to the performance of C . thermocellum. J Am Vet Med Assoc, 1982 Dec 1, 181(11), 1288 - 91 Serologic survey for certain zoonotic diseases in black bears in California; Ruppanner R et al.; Black bears (Ursus americanus) from 3 geographic areas of California were tested for antibodies against agents of 6 zoonotic diseases: toxoplasmosis (indirect hemagglutination), Q fever (microagglutination), trichinosis (latex particle agglutination), botulism (passive hemagglutination), leptospirosis (plate agglutination), and plague (enzyme-linked immunosorbent assay) . Of 149 sera tested, 40 (27%) were positive for Toxoplasma gondii antibodies and 25 (17%) had antibodies against Coxiella burnetii . Of 141 bears tested for Trichinella spiralis, 18 (13%) were seropositive, and 19 (15%) of 125 tested had antibodies against the plague organism, Yersinia pestis . Only 2% (2 of 123 tested) had antibodies against Clostridium botulinum . Sera from 129 bears were tested against 4 pools of Leptospira interrogans representing 12 serovars, and 16% of the sera reacted with the pool containing the serovars australis, hyos, and mini georgia. Mayo Clin Proc, 1982 Dec, 57(12), 737 - 41 Recent experience with antimicrobial susceptibility of anaerobic bacteria: increasing resistance to penicillin; Edson RS et al.; Results of antimicrobial susceptibility testing of anaerobes were reviewed for the last 5 years and compared with two previous surveys from this institution . Allowing for differences in methodology, there appears to be a striking increase in penicillin resistance by the "non-fragilis" Bacteroides . Penicillin concentrations of 50 microgram/ml were required to inhibit 70% of 43 strains tested . The clinical implications of this observation are not known, but isolated reports of therapeutic failure when penicillin was used against the non-fragilis Bacteroides have appeared . Penicillin resistance among Clostridium other than C . perfringens was also noted in the current study . Several strains of clindamycin-resistant Bacteroides fragilis have been observed during the last 2 years, and whether this represents a true increase in resistance will require close scrutiny of clindamycin susceptibility in the future . Future surveillance of antimicrobial susceptibility of anaerobic bacteria will be useful in the detection of any major changes in susceptibility profiles . This knowledge will potentially affect the choice of antimicrobial agent in patients with serious infections caused by anaerobes. J Hyg (Lond), 1982 Dec, 89(3), 507 - 11 Clostridium botulinum type C in the Mersey estuary; Smith GR et al.; Nineteen of 98 samples of mud or sand taken from the Mersey estuary in 1981 contained Clostridium botulinum type C, the organism almost always responsible for botulism in water birds . In the Dungeon and Score Bank areas, where many dead and dying birds were found during the period September-December 1979, almost half the samples contained type C . Most of the positive samples were essentially muddy rather than sandy . The findings do not prove that botulism contributed to the 1979 mortality but are nonetheless thought-provoking, particularly because type C--unlike type B--is by no means ubiquitous in Britain . Type B was present in 12.2% of samples from the Mersey estuary. J Infect Dis, 1982 Dec, 146(6), 791 - 6 Resistance of mice with limited intestinal flora to enteric colonization by Clostridium botulinum; Wells CL et al.; Infant botulism is an age-dependent illness that is caused by the toxin produced by Clostridium botulinum infecting the intestinal tract . Because of composition of the intestinal microflora determines the resistance of mice to enteric colonization by C . botulinum, attempts were made to identify the kinds of bacteria that prevent this in vivo growth . Orogastric challenges of 50 spores of C . botulinum type A were given to adult germfree mice, which are highly susceptible, and to gnotobiotic adult mice carrying the eight species comprising the Charles River Altered Schaedler flora or two or three of the limited number of species constituting a different flora (the University of Wisconsin Gnotobiote Laboratory {UW-GL} flora) . These floras did not prevent infection due to C . botulinum; however, death rates among the mice with defined floras were significantly lower than those among germfree mice exposed to C . botulinum . Botulinum toxin continued to be produced while animals surviving nearly lethal cases of botulism convalesced slowly but uneventfully . Gnotobiotic mice with the complete UW-GL flora were not infected when challenged with 10(5) spores of C . botulinum. J Gen Microbiol, 1982 Dec, 128 (pt 12), 3025 - 35 Benzylpenicillin-induced filament formation of Clostridium perfringens; Williamson R et al.; Growth of Clostridium perfringens with low concentrations of benzylpenicillin inhibited septum formation and division of the organisms . This resulted in continued growth of the organisms as aseptate filaments . The effect was reversed on removal of the antibiotic . The composition of walls isolated from organisms grown with the antibiotic was similar to that of walls from untreated bacteria . In addition, both contained non-N-acetylated glucosamine residues in their peptidoglycan . No differences were detected in the degree of cross-linkage of peptidoglycan . Clostridium perfringens contains six membrane-associated penicillin-binding proteins (PBPs) which have different affinities for {3H}benzylpenicillin . Concentrations of the antibiotic which were sufficient to cause filamentation of apparently all organisms in a culture caused almost complete saturation of PBPs 3, 4, 5 and 6 . At these concentrations there was no measurable interaction with PBPs 1 and 2 . Thus interaction of the antibiotic with the lower molecular weight PBPs is correlated with the inhibition of septum formation in C . perfringens. Infect Immun, 1982 Dec, 38(3), 860 - 4 Selective cytotoxicity of Clostridium perfringens delta toxin on rabbit leukocytes; Jolivet-Reynaud C et al.; Clostridium perfringens delta toxin was selectively cytotoxic for various rabbit leukocyte populations . The sensitivity of these populations to the toxin varied, depending on the tissue from which they were derived, from 28% (appendix) to 41% (bone marrow) and 32% (spleen) . Macrophages were uniformly killed by delta toxin, whereas thymocytes were essentially resistant . Selective cytotoxicity was correlated to the specific binding of the radiolabeled toxin by target cells . The relationship between sensitivity to the toxin and the presence of GM2 ganglioside in the cell membrane of rabbit leukocytes is discussed . Delta toxin might prove a useful new tool for separating leukocyte subpopulations based on their differential sensitivity to the cytolethal effect of this protein. J Clin Pathol, 1982 Dec, 35(12), 1361 - 5 The potential of bacteriocin typing in the study of Clostridium perfringens food poisoning; Watson GN et al.; A range of 49 bacteriocins was used to type 311 strains of Clostridium perfringens isolated from food poisoning outbreaks . Strains of same serotype within an outbreak showed similar patterns of susceptibility to bacteriocins, whereas strains of different serotype isolated from different sources produced many variations in bacteriocin susceptibility patterns . The 311 strains, along with isolates from a wide range of sources were screened for their ability to produce bacteriocins . A much greater proportion of the strains from food poisoning outbreaks was bacteriocinogenic than were isolates from human and animal infections, various foods and the environment. Cell Biophys, 1982 Dec, 4(4), 261 - 71 Effect of purified phospholipases on glucose transport, insulin binding, and insulin action in isolated rat adipocytes; Wieringa T et al.; The influence of alterations in phospholipid structure by phospholipase treatment on insulin action and glucose transport in rat adipocytes was studied . It appeared that phospholipase A2 from bee venom caused a breakdown of approximately 50% of phosphotidylcholine without lysis of the cells . Because of this treatment, insulin binding was increased, resulting in an increased sensitivity of glucose transport towards lower insulin concentrations . Moreover, an increased affinity of the transport system for 2-deoxyglucose was observed . Phospholipase C from Clostridium welchii caused complete lysis of adipocytes . Phospholipase A2 from Crotalus adamenteus was without effect. Biochim Biophys Acta, 1982 Nov 30, 699(2), 92 - 7 Selenium-containing tRNAs from Clostridium sticklandii . Cochromatography of seleno-tRNA I with L-VALYL-tRNA; Chen CS et al.; It has been previously shown that Clostridium sticklandii specifically synthesized three readily separable 75Se-labeled tRNAs, designated seleno-tRNAs I, II and III, and the partially purified seleno-tRNA II cochromatographed with L-prolyl-tRNA on DEAE-Sephadex A-50 (Chen, C.S . and Stadtman, T.C . (1980) Proc . Natl . Acad . Sci . U.S.A . 77, 1403-1407) . In the present study a highly purified 75Se-labeled tRNA I was obtained by chromatography on benzoylated DEAE-cellulose, DEAE-Sephadex A-50 and Sepharose 4B . The 75Se-labeled tRNA I cochromatographed with an L-valine-accepting species on DEAE-Sephadex A-50 and Sepharose 4B . Addition of a 285-fold molar excess of unlabeled L-valine to the L-valine acceptor activity assay mixture markedly decreased the amount of L-{14C}valine bound to seleno-tRNA I. FEBS Lett, 1982 Nov 22, 149(1), 141 - 6 A photo-CIDNP study of the active sites of Megasphaera elsdenii and Clostridium MP flavodoxins; Moonen CT et al.; Megasphaera elsdenii and Clostridium MP flavodoxins have been investigated by photo-CIDNP techniques . Using time-resolved spectroscopy and external dyes carrying different charges it was possible to assign unambiguously the resonance lines in the NMR-spectra to tyrosine, tryptophan and methionine residues in the two proteins . The results show that Trp-91 in M.elsdenii and Trp-90 in Cl.MP flavodoxin are strongly immobilized and placed directly above the benzene subnucleus of the prosthetic group . The data further indicate that the active sites of the two flavodoxins are extremely similar. Minerva Med, 1982 Nov 3, 73(42), 2959 - 63 {Considerations on collateral treatments used with hyperbaric oxygenation in the therapy of gas gangrene}; Gaietta T et al.; The devastating nature of gas gangrene has in the past necessitated radical surgery to prevent the destructive spread of the disease . The advent of antibiotics only partly reduced the need for radical amputations and distressing multilations . Immediate, extensive surgery has now been reduced to a minimum thanks to Hyperbaric Oxygen which rapidly dominates the toxicity of Clostridium . Experience shows that it is still, unfortunately, necessary to emphasize the futility of certain ill-timed and often exaggeratedly multilating surgical approaches, which have at times utterly nullified the clearly positive results obtained by HOT in three cases of post-traumatic gas gangrene. Appl Environ Microbiol, 1982 Nov, 44(5), 1030 - 4 Specificity of bile salt sulfatase activity from Clostridium sp . strains S1; Huijghebaert SM et al.; Clostridium sp . strain S1, an unnamed bile acid-desulfating strain from rat intestinal microflora (S.M . Huijghebaert, J . A . Mertens, and H . J . Eyssen, Appl . Environ . Microbiol . 43:185-192, 1982), was examined for its ability to desulfate different bile acid sulfates and steroid sulfates in growing cultures . Clostridium sp . strain S1 desulfated the 3 alpha-monosulfates of chenodeoxycholic, deoxycholic, and cholic acid, but not their 7 alpha- or 12 alpha-monosulfates . Among the 3-sulfates of the 5 alpha- and 5 beta-bile acids, only bile acid-3-sulfates with an equatorial sulfate group were desulfated . Hence, Clostridium sp . strain S1 desulfated the 3-sulfates of bile acids with a 3 alpha, 5 beta-, a 3 beta, 5 alpha- or a 3 beta, delta 5-structure . In contrast, the bile acid-3-sulfates with a 3 beta, 5 beta- or a 3 alpha, 5 alpha-structure were not desulfated . In addition, Clostridium sp . strain S1 did not hydrolyze the equatorial 3-sulfate esters of C19 and C21 steroids and cholesterol or the phenolic 3-sulfate esters of estrone and estradiol . 23-Nordeoxycholic acid with a C-23 carboxyl group was also not desulfated, in contrast to the 5 beta-bile acid 3 alpha-sulfates with a C-24 or C-26 carboxyl group . Therefore, the specificity of the sulfatase of Clostridium sp . strain S1 is related to the location of the sulfate group on the bile acid molecule, the equatorial orientation of the sulfate group, and the structure of the C-17 side chain, its carboxyl group, and chain length. J Bacteriol, 1982 Nov, 152(2), 946 - 9 Interconversion of valine and leucine by Clostridium sporogenes; Monticello DJ et al.; Clostridium sporogenes has been found to require L-leucine and L-valine for growth in a minimal medium, although valine can be replaced by isobutyrate and leucine by isovalerate . Cells grown in minimal media incorporated significant 14C from {14C}valine into leucine and from {14C}leucine into valine . Growth with {4,5-3H}leucine also resulted in the incorporation of 3H into valine . These results indicate that these bacteria can interconvert leucine and valine. Biochimie, 1982 Nov-Dec, 64(11-12), 1009 - 14 Purification and characterization of a heat stable ferredoxin isolated from Clostridium thermocellum; Forget P; A thermostable ferredoxin was purified from Clostridium thermocellum . The final preparation was homogeneous as judged by electrophoresis in sodium dodecyl sulfate polyacrylamide gel and sedimentation equilibrium . It contains eight atoms of iron and eight acid-labile sulfur groups per molecule, the molecular weight is estimated to be 6 400 and the isoelectric point 3.35 . Its amino-acid composition is characterized by the absence of histidine residues and the presence of eight cysteine residues . The absorption spectrum has a maximum at 390 nm with a molar absorption coefficient of 39 x 10(3) M1 cm-1, similar to that of other bacterial eight iron ferredoxins . The purified ferredoxin has high thermal stability, since the spectrophotometric absorption of the protein at 390 nm did not change after one hour at 70 degrees C and only thirty five per cent of absorbance were lost after one hour at 80 degrees C . With regard to the electron carrier activity, the stability is slightly higher, only twenty five per cent of the activity were lost after one hour at 80 degrees C . During pyruvate oxidation, ferredoxin functions in the transfer of electrons to hydrogenase and also in the back reaction during pyridine nucleotide reduction by a ferredoxin -NAD oxidoreductase using hydrogen as electron donor. Rev Infect Dis, 1982 Nov-Dec, 4(6), 1075 - 95 Antibiotic resistance in anaerobic bacteria: molecular biology and clinical aspects; Bawdon RE et al.; The patterns of antibiotic susceptibility among anaerobes isolated in the United States during the past 14 years were reviewed . Resistance to the tetracyclines, the penicillins, clindamycin, and other antibiotics has emerged among strains of Bacteroides, Clostridium, and anaerobic cocci . Genetic transfer of antibiotic resistance has been documented in anaerobic environments . Inter- and intrageneric transfer by conjugation and transformation has been described . Plasmids have been identified in anaerobes, and conjugal transfer of antibiotic resistance has been reported . The biochemical mechanisms of antibiotic resistance in anaerobes are similar to those described for aerobes . There are some differences in drug transport and inhibitory actions . Antibiotic resistant anaerobes have been isolated from patients participating in large comparative studies of anaerobic infections involving abdominal, pelvic, and pleuropulmonary sites, but instances in which treatment has failed as a result of resistance have not been found . Reports describing small numbers of patients or individual cases have documented the failure of therapy in clinical and laboratory infections caused by both sensitive or resistant anaerobic bacteria . Patterns of antibiotic susceptibility among clinically important anaerobes need to be monitored periodically in several geographic regions. Appl Environ Microbiol, 1982 Nov, 44(5), 1212 - 21 Effects of potassium sorbate and other antibotulinal agents on germination and outgrowth of Clostridium botulinum type E spores in microcultures; Seward RA et al.; The effects of potassium sorbate, sodium hypophosphite, sodium tripolyphosphate, sodium nitrite, and linoleic acid on the germination and outgrowth of Clostridium botulinum type E spores were studied in microcultures . At pH 5.8 to 6.0 in liver veal agar, the germination rate was decreased to nearly zero with 1.0, 1.5, or 2.0% sorbate . At pH 7.0 t 7.2, these levels of sorbate afforded germination and outgrowth of abnormally shaped cells that were defective in cell division . At the high pH range, 0.5 or 1.0% hypophosphite had effects similar to those of sorbate . The use of 0.05% sodium nitrite with sorbate enhanced the lysis of outgrowing cells at pH 7.2 or lower . Emergence and elongation were inhibited by 0.05% linoleic acid with or without 1.0% sorbate at pH 7.0 to 7.2 . The addition of 0.5% tripolyphosphate to media containing 1.5% sorbate at pH 7.1 prevented normal cell growth to an extent greater than with sorbate alone. Am J Med Technol, 1982 Nov, 48(11), 927 - 34 Colitis caused by Clostridium difficile: a review; Weymann LH; Recent evidence has incriminated a toxin-producing anaerobe, Clostridium difficile, as the causative agent of pseudomembranous colitis, an acute inflammatory bowel disease that generally occurs in association with antimicrobial therapy . A wide variety of antimicrobial agents appear to promote C . difficile infection and thereby precipitate colitis . Although the exact pathogenetic mechanisms are not known, several hypotheses, related to the ability of antibiotics to suppress competing bacteria and alter bacterial adhesion to intestinal mucosa, are explored in this review . Laboratory participation in the diagnosis of pseudomembranous colitis caused by C . difficile involves culture and toxin assay . Isolation of C . difficile from feces is facilitated by the use of recently developed selective media . Vancomycin is recommended for treatment of pseudomembranous colitis when removal of the offending antimicrobial agent does not result in clinical improvement. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1982 Nov, 253(2), 265 - 71 {Antibacterial effects of niridazole . II . Effects on aerobic and anaerobic bacteria}; Hof H et al.; Niridazole which is chemically related to metronidazole is endowed with much better antibacterial activity . First, several genera of aerobic bacteria, such as Salmonella and Escherichia, are susceptible to niridazole, whereas metronidazole is completely ineffective . There exist, however, some particular strains which are naturally resistant to niridazole . The in vitro activity of niridazole is still increased, if these facultative anaerobic bacteria are tested under anaerobic growth conditions . Second, niridazole has a higher in vitro activity against anaerobic bacteria, such as Bacteroides spp., Clostridium spp., Fusobacterium spp . and Peptococcus sp., than metronidazole . Extremely low MIC values of niridazole were found ranging from 0.0037 to 0.06 microgram/ml . Propionibacterium acnes, which are resistant to the action of metronidazole, are also relatively resistant to niridazole. Appl Environ Microbiol, 1982 Nov, 44(5), 1144 - 9 Extraction of Clostridium perfringens spores from bottom sediment samples; Emerson DJ et al.; Two extraction-separation procedures were developed and evaluated for use in conjunction with the mCP membrane filter method for the enumeration of Clostridium perfringens spores in bottom sediments . In the more facile of the two procedures, a distilled-water suspension of the sediment sample is pulse sonicated for 10 s and allowed to settle . Portions of the supernatant are then removed for membrane filtration . This procedure is recommended for general use . The more complicated procedure is recommended for situations in which the presence of high levels of toxic materials is suspected or in which relatively low spore densities are present in fine silts . In this procedure, sonication is followed by a distilled water wash . The centrifuged sediment is resuspended in distilled water and mixed with the components of a two-phase separation system (50% polyethylene glycol in distilled water and 25% sucrose in 3 M phosphate buffer {pH 7.1}) . After equilibration of the system and low-speed centrifugation, the top phase and interphase are removed, mixed, and membrane filtered . The recoveries of C . perfringens spores by the two procedures, when used in conjunction with the mCP method, were comparable to each other and significantly greater than those by the British most-probable-number method . It was estimated that more than 85% of the spores were recovered by the procedures . The precision of the sonicate-and-settle-mCP procedure was markedly better than that obtained theoretically by the most-probable-number method and approached that theoretically attributable to counting an average of 85 colonies on each of two plates. J Med Microbiol, 1982 Nov, 15(4), 503 - 9 Isolation and properties of metronidazole-resistant mutants of Clostridium perfringens; Sindar P et al.; Clostridium perfringens strains resistant to metronidazole and tinidazole were isolated from the sensitive parent strain CM288 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine . Strain CM288 was already resistant to rifampicin and nalidixic acid; these genetic markers helped to confirm the identity of mutants . All mutants showed similar characteristics: they grew more slowly than the parent strain and failed to reach the same maximum turbidity; uptake of metronidazole and tinidazole from culture fluids was slow and end products of glucose metabolism were different from those of the parent . Pyruvate dehydrogenase activity was not detected in broken cell preparations of the mutant strains although this enzyme was readily detected in the parent strain . Changes in end products of glucose metabolism were consistent with the absence of pyruvate dehydrogenase activity because pyruvate was accumulated during growth and lactate levels were higher whereas acetate, CO2 and ethanol levels were diminished. Infect Immun, 1982 Nov, 38(2), 592 - 7 Purification of two Clostridium perfringens enterotoxin-like proteins and their effects on membrane permeability in primary cultures of adult rat hepatocytes; Dasgupta BR et al.; We isolated two proteins, ET-1 and ET-2, from the sporangial extracts of Clostridium perfringens type A . Both proteins had some properties in common with the well-known C . perfringens enterotoxin . ET-1 and ET-2 behaved as single and distinct entities in anion exchange chromatography and disk gel electrophoresis . ET-2 was the more anionic protein since it eluted more slowly from the anion exchange column and migrated faster toward the anode in polyacrylamide disk gel electrophoresis (pH 8.5, native gels) . Additionally, in this electrophoretic system ET-2 was not distinguishable from the enterotoxin . The amino acid compositions of ET-1 and ET-2 were similar but differed in a few amino acid residues . The values for both proteins were also similar to the published reports of others for the enterotoxin . Both ET-1 and ET-2 showed lines of identity in agar gel double immunodiffusion against anti-enterotoxin antiserum . Both ET-1 and ET-2 were toxic for rat hepatocytes in primary monolayer culture as determined by accelerated exodus of L-{14C}glucose from preloaded cells and by the rapid uptake of 45Ca2+ after exposure to the proteins . In this regard, ET-1 and ET-2 appeared to be identical in mechanism of action to what has been regarded in the literature as "the" C . perfringens enterotoxin . Interestingly, ET-2 was 3 to 10 times more toxic on a weight basis than ET-1 was. Pharmacotherapy, 1982 Nov-Dec, 2(6), 287 - 99 Piperacillin sodium: antibacterial spectrum, pharmacokinetics, clinical efficacy, and adverse reactions; Fortner CL et al.; Piperacillin sodium is a beta lactam antibiotic with a broad range of antibacterial activity that includes gram-negative bacilli, gram-positive cocci (except penicillinase-producing S . aureus) and anaerobic pathogens such as Clostridium difficile, and Bacteroides fragilis . Piperacillin inhibits many of the members of the Enterobacteriaceae, including Klebsiella sp and Pseudomonas, at lower concentrations than required for carbenicillin and ticarcillin . Piperacillin sodium is administered by intramuscular and intravenous injection and is widely distributed throughout body fluids and tissues . Like other newer penicillins, piperacillin is excreted by both renal and biliary mechanisms . The primary route of elimination is by glomerular filtration, which results in high urinary concentrations of the unchanged compound . Piperacillin has been approved for patients with serious infection caused by susceptible strains of specific organisms in intra-abdominal, urinary tract, gynecologic, lower respiratory tract, skin and skin structure, bone and joint, and gonococcal infections and septicemia . As with other penicillins, piperacillin has a low frequency of toxicity . The usual dose of piperacillin in adults with serious infections with normal renal function is 3-4 g every 4-6 hr as a 20-30 min infusion, with a maximum dose of 24 g per day . It is stable in most large volume parenteral solutions . Less serious infectins (requiring smaller dosages) may be treated by intramuscular injection; however, no more than 2 g should be given at any one injection site . Overall, piperacillin has a greater degree of activity than other penicillins . Evidence from prospective studies indicates that piperacillin is a highly effective agent for the treatment of patients with infections caused by susceptible organisms. Rev Infect Dis, 1982 Nov-Dec, 4 Suppl, S511 - 5 In vitro activity of moxalactam against anaerobic bacteria; Gilchrist MJ et al.; Moxalactam was tested against 80 isolates of anaerobic bacteria from clinical specimens . The results of this study and a review of other studies, in which comparable susceptibility testing methods were used, demonstrated the susceptibility to moxalactam of members of the Bacteroides fragilis group, Bacteroides melaninogenicus, Fusobacterium species, anaerobic gram-positive cocci, and Clostridium perfringens. Arch Intern Med, 1982 Oct 25, 142(11), 1988 - 92 Pathogenic anaerobes; Finegold SM; Anaerobes are prevalent on all mucosal surfaces and virtually all anaerobic infections are endogenous . Two thirds of anaerobic infections involve five anaerobic organisms or groups--the Bacteroides fragilis group, the Bacteroides melaninogenicus-Bacteroides asaccharolyticus group, Fusobacterium nucleatum, the anaerobic cocci, and Clostridium perfringens . Conditions that lower the oxidation-reduction potential and disrupt the mucosal surface (eg, vascular problems, malignant neoplasms, and surgery) lead to infection with anaerobes . Clues to anaerobic infection include foul odor, gas, tissue destruction, underlying malignant neoplasms, and the unique appearance of certain anaerobes on Gram's stain . Specimens must be collected to avoid normal flora and transported to the laboratory under anaerobic conditions . Therapy involves surgical debridement and drainage and the use of various antimicrobial agents . Antimicrobial agents must be used for extended periods to avoid relapse. JAMA, 1982 Oct 22, 248(16), 2028 - 9 Implication of plasma free hemoglobin in massive clostridial hemolysis; Terebelo HR et al.; Prolonged tissue oxygenation and maintenance of intravascular oncotic pressure are severely impaired with the absence of an effective RBC mass . We recently encountered a patient with a nondetectable hematocrit value (0%) attributed to Clostridium perfringens hemolysis . The patient maintained normal BP, tissue oxygenation, and mentation and survived longer than four hours, despite ineffective transfusion therapy . Plasma free hemoglobin was responsible for the preservation of tissue oxygenation, intravascular oncotic pressure, and pH . Existing studies in animals support stroma-free hemoglobin as an effective blood product in humans. Biochim Biophys Acta, 1982 Oct 20, 708(1), 6 - 11 Inhibition of protein synthesis by a tryptic polypeptide of Clostridium perfringens type A enterotoxin; Granum PE; The biological activity of Clostridium perfringens enterotoxin can be tested more precisely and with a much higher sensitivity by using the inhibition of protein synthesis by Vero cells, rather than the guinea pig skin test . Tryptic peptides of the enterotoxin produced in the presence of different concentrations of sodium dodecyl sulfate (0-1%) have been tested for biological activity (Vero cells) and inhibitory effect on cell-free protein synthesis (rabbit reticulocyte lysate) . A fraction of tryptic peptides, about 16,000 daltons, was able to inhibit the cell-free protein synthesis, while the native enterotoxin had no such effect . The 16 kDa fraction had, however, lost the ability to disrupt the Vero cells (normal biological activity) . It is probable that the enterotoxin has the double function (A and B chain), known from several other toxins, confined in its single polypeptide chain. C R Seances Acad Sci III, 1982 Oct 4, 295(4), 299 - 302 {Neutral and basic compounds present in gases produced by Clostridium sporogenes, Plectridium putrificum and Plectridium glycolicum cultured under vacuum in sodium thioglycolate glucose broth}; Rimbault A et al.; The study of gases produced by Clostridium sporogenes, Plectridium putrificum et P . glycolicum cultured under vacuum in sodium thioglycolate glucose broth makes possible the detection of compounds not described before for these Bacteria, particularly methyl-2 butanal and methyl-3 butanal for P . glycolicum and thiacyclopropane in these 3 Bacteria . Thiacyclopropane is the result of the cyclization of mercapto-2 ethanol produced by the reduction of thioglycolic acid. J Clin Microbiol, 1982 Oct, 16(4), 659 - 62 Isolation of Clostridium difficile from hospitalized patients without antibiotic-associated diarrhea or colitis; Varki NM et al.; Stool samples from 100 hospitalized patients and 21 healthy adults, obtained between March and June 1980, were cultured on a special selective medium containing cefoxitin and cycloserine to detect Clostridium difficile . This organism was isolated from 13 of the hospitalized patients and from 1 healthy subject . None of the patients with positive cultures had received antimicrobial therapy in the 3 preceding months . The observed rate of C . difficile isolation from adults not suffering from antibiotic-associated diarrhea or colitis is higher than previously reported . C . difficile culture is not recommended as a substitute for toxin assay in the evaluation of patients with intestinal disorders after antimicrobial chemotherapy. J Clin Microbiol, 1982 Oct, 16(4), 637 - 40 Survey of the extrachromosomal gene pool of Clostridium difficile; Muldrow LL et al.; Pseudomembranous colitis, a severe diarrheal disease, has been linked to the administration of antibiotics and to two toxins produced by Clostridium difficile . Eighty-two strains of C . difficile isolated from humans and hamsters were assayed for the presence of plasmid DNA . Agarose gel electrophoresis of Sarkosyl-lysed cells indicated that 18% of the strains contained from one to four plasmids . The plasmid DNA in these strains ranged in molecular weight from 2.7 X 10(6) to 60 X 10(6) . Strains with and without plasmids were examined for the cytopathogenic effect of the toxins on MRC-5 cells . No correlation was observed between plasmid content and cytopathogenic effect . The results of in vitro antibiotic susceptibility testing with plasmid-containing strains revealed that 33% of the strains tested exhibited growth with four or more of the antimicrobial agents used. Hoppe Seylers Z Physiol Chem, 1982 Oct, 363(10), 1253 - 7 The stereochemical course of the water elimination from (2R)-phenyllactate in the amino acid fermentation of Clostridium sporogenes; Pitsch C et al.; (2R,3R)-{3-3H}- and (2R,3S)-{3-3H}- phenyllactate were synthesized from phenylpyruvate with the help of phenylpyruvate tautomerase and an NADH-dependent 2-oxo-acid reductase from Clostridium sporogenes . With whole cells of C . sporogenes both (2R)-phenyllactates are mainly transformed to phenylpropionate and, to a minor extent to phenylalanine . According to recently published results, the former transformation leads from phenyllactate, or an activated derivative of it, to (E)-cinnamate which is reduced to phenylpropionate in a fast consecutive reaction . The 3H/14C-ratio of phenylpropionate deriving from (2R,3R)-{3-3H,U-14C}phenyllactate was about 85% of that of the phenyllactate and the corresponding ratio for phenylpropionate deriving from the (2R,3S)-{3-3H,U-14C}phenyllactate about 7%, respectively . Assuming that the E-configuration of cinnamate is formed directly from (2R)-phenyllactate, the water elimination occurs in a syn fashion. Gastroenterology, 1982 Oct, 83(4), 836 - 43 Myoelectric effects of Clostridium difficile: motility-altering factors distinct from its cytotoxin and enterotoxin in rabbits; Justus PG et al.; Clostridium difficile is a bacterium that causes antibiotic-associated pseudomembraneous enterocolitis . This bacterium produces a cytotoxin that induces tissue culture assay positivity and an enterotoxin that causes in vivo mucosal injury . In previous studies we have described two altered myoelectric patterns in response to certain diarrheagenic organisms in an in vivo rabbit model . The first pattern was called the migrating action potential complex and is associated with noninvasive agents; the second pattern was called repetitive bursts of action potentials and is characteristic of invasive or cytolytic agents . In this study, we evaluated the effects of purified cytotoxin (2.5-3.75 micrograms) and enterotoxin (140 micrograms) from C . difficile on the myoelectric activity in isolated ileal loops in New Zealand White rabbits . These observations in myoelectric activity were correlated with the results of similar studies by using the crude culture filtrates from C . difficile, or the products of Amicon XM50 filtration of its culture supernatant resulting in a high molecular weight product (0.3 mg protein/ml) and a low molecular weight product (0.57 mg protein/ml) . Monopolar silver-silver chloride electrodes were used to record all myoelectric activity for an 8-h period . The animals were then killed, and tissue obtained from the ileal loops was histologically evaluated . Crude culture filtrates of C . difficile induced 7.0 migrating action potential complexes/hour and 6.8 repetitive bursts of action potentials/hour . Saline controls induced no migrating action potential complexes and 0.1 repetitive bursts of action potentials/hour . The high molecular weight filtration product obtained from the culture supernatant of C . difficile induced significantly more repetitive bursts of action potentials (41.1/h) than all agents studied . The purified cytotoxin or enterotoxin induced no migrating action potential complex activity and minimal repetitive bursts of action potential activity (0.9/h and 0.6/h, respectively) . These values were not different from the saline controls; however, only the enterotoxin and the high molecular weight filtration product caused mucosal damage . These studies suggest that C . difficile produces a heat-labile substance or substances that alter the motility of the small intestine independent of the proteins responsible for in vivo tissue damage and cytotoxin assay positivity. Aust N Z J Med, 1982 Oct, 12(5), 535 - 7 Reactive arthritis associated with Clostridium difficile; McCluskey J et al.; A case of reactive arthritis in a patient with a previously documented history of Reiter's syndrome is described . The precipitating agent appears to have been Clostridium difficile . High levels of toxin were demonstrable in the faeces and neutralising antitoxin was detected in the patient's serum but not synovial fluid . Resolution of the polyarthropathy was slow despite successful eradication of the C . difficile with a course of vancomycin. Mol Cell Biochem, 1982 Oct 1, 48(1), 25 - 32 Clostridium botulinum type C toxin: a sketch of the molecule; Syuto B et al.; The purification and crystallization of type C botulinum toxin along with its physical characteristics are described . The shape of Clostridium botulinum type C toxin molecule is globular like a pressed ball with a 7.4 nm diameter and a 4.3 nm thickness . The molecular volume is approximately 185 nl and the molecular weight is 141000 . The toxin molecule is composed of two parts, which are separable under appropriate conditions . These parts have some differences in the electrophoretic properties, amino acid distribution, immunological, and functional characteristics . The toxin molecule can be reconstituted by association of S-S bond between the two chains . The expression of the toxicity requires that the fragments of the polypeptide chain carrying the necessary information be functionally organized for the proper development of the specific tertiary structure for active conformation. Jpn J Med Sci Biol, 1982 Oct-Dec, 35(5-6), 239 - 42 Experimental toxicoinfection in infant mice challenged with spores of Clostridium botulinum type E; Mitamura H et al.; Conventionally raised suckling mice were given 10(7) spores of a strain of Clostridium botulinum type E . Most but not all infant mice aged 8 through 19 days at the time of administration died after developing symptoms typical of botulism . However, none of the infant mice challenged with the spores at dose levels lower than 10(6) spores/mouse developed illness. Jpn J Med Sci Biol, 1982 Oct-Dec, 35(5-6), 203 - 11 The relation between toxicity and toxin-related-antigen contents of Clostridium botulinum types C and D cultures as determined by mouse bioassay and ELISA; Notermans S et al.; ELISA was tested for the adequacy to differentiate between botulinum type C1 and D toxins . The results presented in this paper indicate antigenic relationship between type C1 and D toxins . Furthermore, indications were obtained that the antigenicities of type C1 and of D toxins produced by different strains are not identical . Although ELISA can be used to differentiate type C1 and D toxins from those of other types, the assay has only limited value to differentiate between type C1 and D toxins . No parallel relationship between toxicity (mouse bioassay) and toxin-related-antigen contents (ELISA) of C . botulinum type C and D cultures was found. Zh Mikrobiol Epidemiol Immunobiol, 1982 Oct, (10), 43 - 7 {Fine morphology of the anomalies of sporulation in Clostridium tetani}; Vaisman ISh; The electron microscopic study of the cultures of Cl . tetani strains Nos . 154 and 741 has been carried out, starting from the late stationary phase of their development in a liquid culture medium prepared on the basis of casein hydrolysate . Under experimental conditions sporogenesis is observed in 17-33% of the organisms in the population of strain No . 154 and in 7-14% of the organisms in the population of strain No . 471 . In most of the sporulating cells sporogenesis occurs in consecutive stages commonly observed in clostridia . Anomalies in the formation of pores are manifested by the ectopic initial formation of protospores, the disordered excess growth of osmiophil structures of the exosporic type, the early lysis of the cytoplasmic structures of the sporangium cell or the spore itself . Such changes resulting in the death of the cell are regarded as the pathomorphology of the bacterial cell. J Clin Pathol, 1982 Oct, 35(10), 1158 - 62 The use of an anaerobic incubator for the isolation of anaerobes from clinical samples; Berry PL et al.; An anaerobic incubator was compared with a standard jar system for the isolation of anaerobes from clinical material . Seventy specimens were selected as likely to yield anaerobes: 342 different anaerobes were isolated in the incubator and 347 in anaebrobic jars . These included Bacteroides spp (43%), Peptococcus spp (26%), Peptostreptococcus spp (13%), Veillonella spp (7%), Fusobacterium spp (7%), Clostridium spp (2%) and miscellaneous Gram-positive nonsporing bacilli (2%) . Differences in isolation rates for each system were inconsistent and minor . Sixteen anaerobes were chosen for quantitative tests at the beginning and end of the study period . Miles and Misra counts showed a slight advantage of the incubator for F nucleatum, but no difference for B fragilis, B thetaiomicron, B uniformis, B bivius, B corrodens, F mortiferum, Ps anaerobius, P prevotii or Propionibacterium acnes . In almost all cases, colonies in anaerobic jars were slightly larger than those in the incubator . Disc antibiotic sensitivity tests gave the same results in each system, at the beginning and end of the study period . The anaerobic incubator provides an effective means of isolation of anaerobes in a clinical laboratory . However, several design features of the prototype would require change if the system were introduced. J Clin Microbiol, 1982 Oct, 16(4), 761 - 2 Clinical laboratory evaluation of a reverse CAMP test for presumptive identification of Clostridium perfringens; Buchanan AG; Ninety-six percent of Clostridium perfringens isolates from clinical specimens were reverse CAMP test positive, whereas several other Clostridium species tested were reverse CAMP test negative . C . perfringens was detected by direct inoculation of clinical specimens to reverse CAMP plates, and the reverse CAMP procedure provided reliable presumptive identification of this organism. Acta Pathol Microbiol Immunol Scand {B}, 1982 Oct, 90(5), 377 - 81 Binding of Clostridium perfringens enterotoxin to hepatocytes, small intestinal epithelial cells and Vero cells; Jarmund T et al.; Clostridium perfringens enterotoxin binds specifically to hepatocytes, Vero cells and rat intestinal epithelial cells, and causes membrane damage visible as "bubble" formation . Fluorescein-labelled enterotoxin also binds specifically to the same cell types . 125I labelled enterotoxin shows one class of binding sites on both Vero and rat intestinal epithelial cells . The number of binding sites decreases along the rat intestine, but the affinity constant is the same in different parts of the intestine. Infect Immun, 1982 Oct, 38(1), 386 - 8 Clostridium perfringens type A enterotoxin: characterization of the amino-terminal region; Duffy LK et al.; The amino-terminal region of the enterotoxin of Clostridium perfringens was investigated by automated sequence analysis . The primary structure results revealed that the enterotoxin is composed of a single polypeptide amino acid sequence . Computer comparison of a 20-residue sequence with a sequence library of reported proteins revealed no significant chemical similarities, indicating that the enterotoxin represents a unique polypeptide primary structure. Infect Immun, 1982 Oct, 38(1), 14 - 20 Four different monoclonal antibodies against type C1 toxin of Clostridium botulinum; Oguma K et al.; Monoclonal antibodies against type C1 toxin produced by Clostridium botulinum type C strain Stockholm (C-ST) were prepared by fusion of BALB/c myeloma cells P3X63-Ag8, with spleen cells from the mice immunized by C-ST toxoid . About 5% of single-cell colonies in wells were found to produce antibodies against the toxin as determined by an enzyme-linked immunosorbent assay (ELISA) . Four different hybridoma cell lines, no . 9, 12, 14, and 17, were established, cloned by limiting dilution, and intraperitoneally injected into mice to obtain the ascites fluids containing high-titered antibodies . The reactions of these antibodies to type C1 and D toxins of strains C-ST, D-1873, and D-South African (D-SA) were observed by both neutralization and ELISA tests . Three monoclonal antibodies, no . 9, 14, and 17, reacted with C-ST toxin, but only no . 17 highly neutralized the toxin . These antibodies did not react with type D toxins . On the contrary, no . 12 reacted with toxins of both C-ST and D-SA (but not of D-1873) and commonly neutralized these two toxins . This indicates that there is a common antigenic part between C-ST and D-SA toxin molecules which participates in the toxin-neutralizing reaction . The neutralization profiles of C-ST toxin by no . 12 and 17 antibodies were different in a time-to-death test of mice . The mechanisms of neutralization by no . 12 and 17 may be different. C R Seances Acad Sci III, 1982 Sep 27, 295(3), 219 - 21 {Neutral and basic compounds present in gases produced by Clostridium histolyticum, Clostridium hastiforme and Clostridium ghoni cultured under vacuum in sodium thioglycolate glucose broth}; Rimbault A et al.; The study of gases produced by Clostridium histolyticum, C . hastiforme and C . ghoni cultured under vacuum in sodium thioglycolate glucose broth makes it possible to detect compounds not described before for these Bacteria, and confirms the production of methane in anaerobic spore-forming Bacteria. Biochim Biophys Acta, 1982 Sep 22, 707(1), 147 - 53 Effects of temperature, pH and detergents on the molecular conformation of the enterotoxin of Clostridium perfringens; Salinovich O et al.; The effects of temperature, pH and sodium dodecyl sulfate on the conformation of the enterotoxin from Clostridium perfringens type A were followed by circular dichroism in both the peptide and aromatic regions . At near-physiological conditions (35 degrees C, pH 6.7) the enterotoxin exhibited a conformation consisting of approximately 60% pleated sheet, 40% non-periodic, and essentially no helix . The peptide region was relatively stable at temperatures up to 55 degrees C and at pH values ranging from 4-10 . The aromatic region demonstrated profound, time-dependent changes at 55 degrees C . At temperatures greater than 55 degrees C, extremes of pH, and in the presence of SDS, the spectra in both regions showed major structural reorganization; in most cases a gain in helical content at the expense of sheet structure was observed . The conformational properties of the protein are very similar to those observed for the lectins, a group of carbohydrate-binding proteins. Biochemistry, 1982 Sep 14, 21(19), 4762 - 71 Characterization of the selenium-substituted 2 {4Fe-4Se} ferredoxin from Clostridium pasteurianum; Moulis JM et al.; The sulfur atoms of the two {4Fe-4S} clusters present in the ferredoxin from Clostridium pasteurianum have been replaced by selenium . The substitution is readily carried out by incubating the apoferredoxin with excess amounts of Fe3+, selenite, and dithiothreitol under anaerobic conditions . The UV-visible absorption spectrum of the Se-substituted ferredoxin, the core extrusion of its active sites, and analyses of its iron and selenium contents show that it contains two {4Fe-4Se} clusters . The Se-substituted ferredoxin is considerably less resistant to oxygen or to acidic and alkaline pH than the native ferredoxin: the half-lives of the former are 20-500 times shorter than those of the latter . The native ferredoxin and the Se-substituted ferredoxin display similar kinetic properties when used as electron donors to the hydrogenase from C . pasteurianum . It is of note, however, that the Km and Vmax values are lower for the 2{4Fe-4Se} ferredoxin than for the 2{4Fe-4S} ferredoxin . Reductive and oxidative titrations with dithionite and with thionine, respectively, show that both ferredoxins are two-electron carriers . The redox potentials of the ferredoxins have been measured by equilibrating them with the H2/H+ couple via hydrogenase: values of -423 and -417 mV have been found for the 2{4Fe-4S} ferredoxin and 2{4Fe-4Se} ferredoxin, respectively . Ferredoxins containing both chalcogenides in their {4Fe-4X} (X = S, Se) clusters have been prepared by reconstitution reactions involving mixtures of sulfide and selenide: the latter experiments show that sulfide and selenide are equally reactive in the incorporation of {4Fe-4X} (X = S, Se) sites into ferredoxin . The present report, together with former studies, establishes the general feasibility of the Se/S substitution in {2Fe-2S} and in {4Fe-4S} clusters of proteins and of synthetic analogues. Steroids, 1982 Sep, 40(3), 347 - 57 Antibacterial properties of bile acid oxazoline compounds; Hylemon PB et al.; Chenooxazoline (50-100 microM) inhibited (greater than 50%) both 7 alpha and 7 beta-dehydroxylase activities in whole cells and cell extracts of Eubacterium sp . V.P.I . 12708 . Chenooxazoline (greater than or equal to 50 microM) and methylchenooxazoline (greater than 25 microM) but not lithooxazoline (less than or equal to 100 microM) inhibited growing cultures of Eubacterium sp . V.P.I . 12708 . Chenooxazoline (100 microM) also inhibited the growth of certain members of the genera Eubacterium, Clostridium, Bacteroides and Staphylococcus but not Pseudomonas, Escherichia, Salmonella or the eucaryotic microorganism, Saccharomyces cerevisiae (less than or equal to 400 microM). Pediatr Infect Dis, 1982 Sep-Oct, 1(5), 336 - 8 Clostridium difficile in a pediatric outpatient population; Boenning DA et al.; Clostridium difficile has been implicated as one cause of hospital-acquired diarrhea in children, yet the prevalence of this organism in outpatient children with diarrhea has not been established . Over a 1-year period, 306 outpatient children ranging in age from 2 weeks to 16 years were cultured for C . difficile and potential bacterial pathogens . C . difficile was isolated from 7.0% of patients with diarrhea (12 of 171) and 14.8% of controls with nondiarrheal illnesses (20 of 135) . The 32 patients yielding C . difficile were significantly younger than the other study patients . C . difficile was isolated in conjunction with another enteric pathogen in only one case . Antibiotic exposure in the month prior to culture was no different between the 32 positive patients and the overall population . Moreover the patients yielding C . difficile were clinically indistinguishable from the other study patients . C . difficile appears to comprise part of the normal bowel flora in some children beyond the neonatal period and despite a negative history of recent antibiotic usage. Ann Microbiol (Paris), 1982 Sep-Oct, 133(2), 281 - 90 Direct quantitative determination of acidic end products in clinical specimens for presumptive diagnosis of anaerobic infections; Legakis NJ et al.; Acidic metabolic products determined directly in clinical specimens of 51 anaerobic and 12 aerobic infections by quantitative gas-liquid chromatography (GLC) were correlated with the bacteriologic findings . The presence of appreciable amounts of succinic acid, more than 1 microM per ml, propionic and isovaleric acid, at concentrations not exceeding 3 microM per ml, was strong evidence for Bacteroides fragilis infections . When isobutyric acid was also detected, a B . melaninogenicus infection could be presumed . However, the presence of other anaerobes or aerobes could not be excluded . The presence of Clostridium spp . could also be presumed by the detection of butyric acid at a concentration greater than 4 microM per ml and of acetic, propionic and isobutyric acid at relatively high concentrations . In specimens from aerobic infections, no other acid except acetic and lactic acid could be detected . Our data also show that quantitative GLC is not valid in Peptococcus and Peptostreptococcus spp . infections except in some cases where Peptococcus alone or together with aerobic organisms are found. Vet Pathol Suppl, 1982 Sep, 7, 126 - 33 Acute gastric dilatation in nonhuman primates: review and case studies; Pond CL et al.; Acute gastric dilatation occurs sporadically in laboratory-housed nonhuman primates . Clinical histories often include chronic drug administration, food restriction, accidental overfeeding, and prior anesthesia . Monkeys may be found dead or may have clinical signs of colic, abdominal distention, and dyspnea . Death in untreated cases is due to impaired venous return and cardiopulmonary failure . Gastric distention with fermented gaseous ingesta and congestion of the abdominal viscera are the predominant lesions . The cause of acute gastric dilatation is unknown, but it probably is multifactorial . Two principal factors seem to be intragastric fermentation associated with Clostridium perfringens, and abnormal gastric function. Can J Microbiol, 1982 Sep, 28(9), 1032 - 6 Response of Clostridium perfringens and its L form to bacteriocins of C . perfringens; Mahony DE; Clostridium perfringens strain No . 28 and its penicillin-induced stable L form were treated with 10 different bacteriocins of C . perfringens . Viable count and labelled amino acid incorporation experiments revealed that the L form was sensitive to two and possibly three bacteriocins to which the bacillus was not, while both forms were commonly sensitive to two other bacteriocins and resistant to five others . Adsorption of bacteriocin, immunity factors, or perhaps uptake of bacteriocin might be proposed to explain the responses of these organisms to bacteriocins. J Bacteriol, 1982 Sep, 151(3), 1372 - 9 Partial purification and properties of phosphatidylserine synthase from Clostridium perfringens; Cousminer JJ et al.; The membrane-associated phospholipid biosynthetic enzyme cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase; EC 2.7.8.8) was partially purified 337-fold from a cell-free extract of the gram-positive pathogenic anaerobe Clostridium perfringens (ATCC 3624) . The purification procedure included extraction from the cell envelope with the nonionic detergent Triton X-100, followed by affinity chromatography on cytidine 5'-diphosphate-diacylglycerol-Sepharose . When the partially purified enzyme was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two major bands were evident with apparent minimum molecular weights of 39,000 and 31,000 . Activity of phosphatidylserine synthase was dependent on the addition of manganese ions (3 mM) and Triton X-100 (2.7 mM) for maximum activity . The rate of catalysis was maximal at 40 degrees C (with rapid thermal inactivation above this temperature), and the pH optimum was 8.5 . The apparent Km values for cytidine 5'-diphosphate-diacylglycerol and L-serine were 0.24 and 0.26 mM, respectively . The synthetic (forward) reaction was favored, as indicated by an equilibrium constant of 82, and the energy of activation was found to be 18 kcal/mol (75,362 J/mol). Eur J Biochem, 1982 Sep, 127(1), 105 - 16 On the mode of action of the bacteriocin butyricin 7423 . Effects on membrane potential and potassium-ion accumulation in Clostridium pasteurianum; Clarke DJ et al.; 1 . The apparent transmembrane bulk-phase electrical potential (delta psi) of Clostridium pasteurianum was determined from the distribution ratio of the membrane-permeable cation butyltriphenylphosphonium (BuPh3P+) . In glycolysing cells the highest value of delta psi, calculated on the assumption that there was no energy-dependent binding of BuPh3P+ to the organisms, was recorded in media containing only 2-3 mM K+ ions and, even so, was only 100-110 mV . 2 . Efrapeptin, a BF1-directed inhibitor of the membrane H+-ATPase of Cl . pasteurianum, abolished the membrane potential (delta psi) and caused complete efflux of actively-transported K+ ions . Thus protonmotive hydrolysis of ATP generated by substrate level phosphorylation is the sole means of membrane energisation in this anaerobe . 3 . At low (sublethal) concentrations, butyricin 7423 stimulated K+ efflux from Cl . pasteurianum without measurably affecting its membrane potential . At lethal and supralethal concentrations of this bacteriocin, both delta psi and active K+ uptake were abolished . 4 . Whilst the addition of valinomycin to cells of Cl . pasteurianum suspended in media of low K+ concentration generated a diffusion potential to which BuPh3P+ would respond, addition of butyricin 7423 in place of valinomycin caused no such effect . Also, unlike valinomycin, butyricin 7423 did not increase the rate of K+ efflux from non-glycolysing cells of Cl . pasteurianum . Valinomycin stimulated, but butyricin 7423 inhibited, the uptake of 86Rb+ ions by glycolysing cells of Cl . pasteurianum . 5 . A mutant strain of Cl . pasteurianum (viz . strain DC3) which possessed a H+-ATPase with diminished sensitivity both to N,N'-dicyclohexylcarbodiimide and to butyricin 7423, exhibited a negligible decrease in delta psi and in K+ accumulation ratio in response to concentrations of butyricin 7423 that were bactericidal to the wild-type, parent organism . Even so, the bactericidal action of butyricin 7423 on Cl . pasteurianum is not adequately explained by its ability in vitro to inhibit the membrane H+-ATPase of this organism . 6 . Bactericidal concentrations of butyricin 7423 neither provoked efflux of Na+ ions from Cl . pasteurianum nor exhibited any protonophorous activity . However, at artificially high concentration, butyricin 7423 catalysed the passage of Na+ ions as well as of K+ ions through multilayer lipid membranes . 7 . As a non-protonophorous uncoupler, butyricin 7423 appears to act in a similar manner to that of the membrane-active colicins . Yet no evidence was obtained that butyricin 7423 at its minimum lethal concentration might form a gated ion channel in the cytoplasmic membrane of the target cell, or act as a classic ionophore. Vet Pathol Suppl, 1982 Sep, 19 Suppl 7, 126 - 33 Acute gastric dilatation in nonhuman primates: review and case studies; Pond CL et al.; Acute gastric dilatation occurs sporadically in laboratory-housed nonhuman primates . Clinical histories often include chronic drug administration, food restriction, accidental overfeeding, and prior anesthesia . Monkeys may be found dead or may have clinical signs of colic, abdominal distention, and dyspnea . Death in untreated cases is due to impaired venous return and cardiopulmonary failure . Gastric distention with fermented gaseous ingesta and congestion of the abdominal viscera are the predominant lesions . The cause of acute gastric dilatation is unknown, but it probably is multifactorial . Two principal factors seem to be intragastric fermentation associated with Clostridium perfringens, and abnormal gastric function. Thromb Res, 1982 Aug 15, 27(4), 405 - 17 Investigation of the effects of phospholipase C on human platelets: evidence that aggregation induced by phospholipase C is independent of prostaglandin generation, released ADP and is modulated by cyclic AMP; Akbar H et al.; Effects and the mechanism of action of phospholipase C (PLC), from Clostridium perfringens, on washed human platelets were examined to better understand the role of PLC in platelet function . PLC caused aggregation and secretion of {14C}-5HT, without concomitant loss of cytoplasmic, LDH, in a concentration dependent manner . P-nitrophenylphosphorylcholine, a substrate for PLC, blocked these responses in a concentration dependent manner . In other experiments hirudin, alpha-1-antitrypsin and soybean trypsin inhibitor did not inhibit PLC-induced activation of human platelets . PLC-induced aggregation and {14C}-5HT secretion was not inhibited by aspirin, a known inhibitor of prostaglandin biosynthesis . PLC-induced aggregation was selectively inhibited by analogs of 7,8-dihydroxybenzazepine and 7,8-methylenedioxybenzazepine in a concentration dependent manner . These two agents had no effect on arachidonic acid-induced aggregation . PLC-induced aggregation was not inhibited by apyrase, an enzyme which hydrolyzes ADP . In other experiments, PLC-treated platelets did not exhibit any platelet activating factor-like activity . Prostaglandin E1 and trifluoperazine showed concentration dependent inhibitor effects on PLC-mediated aggregation and secretion of {14C}-5HT . These findings indicate that: a) PLC is capable of inducing aggregation and specific secretion of {14C}-5HT without causing lysis of platelets; b) mechanism of PLC-induced activation of platelets is independent of prostaglandin generation or action, released ADP, and PAF; and c) cyclic AMP plays a modulatory role in PLC-mediated secretion and aggregation of human platelets. Gastroenterology, 1982 Aug, 83(2), 465 - 9 Possible foodborne transmission in a case of pseudomembranous colitis due to Clostridium difficile: influence of gastrointestinal secretions on Clostridium difficile infection; Gurian L et al.; A 78-yr-old woman with a history of hypochlorhydria was found to have pseudomembranous colitis due to Clostridium difficile . She had not received previous antimicrobial therapy . Her onset of disease followed ingestion of possibly contaminated canned salmon, suggesting possible oral transmission of disease . We assessed the possibility of ingested Clostridium difficile organisms or cytotoxin surviving passage through the upper gastrointestinal tract . Normal gastric juice, hypochlorhydric gastric juice, and duodenal secretions were obtained from volunteers and tested for their ability to kill Clostridium difficile organisms or inactivate toxin . These in vitro studies indicated that the primary upper gastrointestinal barriers for ingested Clostridium difficile and cytotoxin were pH-dependent . We suggest that oral transmission of disease due to Clostridium difficile may occur in hypochlorhydric patients. Proc Natl Acad Sci U S A, 1982 Aug, 79(16), 4912 - 6 Isolation of a selenium-containing thiolase from Clostridium kluyveri: identification of the selenium moiety as selenomethionine; Hartmanis MG et al.; Clostridium kluyveri grown in the presence of 1 muM Na2(75)SeO3 produces a thiolase that copurifies with 75Se . Based on several criteria, the selenium moiety in this protein is selenomethionine . The 75Se-labeled amino acid in acid hydrolysates of the radioactive protein cochromatographed with authentic selenomethionine on an amino acid analyzer and on TLC plates in acidic and basic solvents . Incubation with S-adenosylmethionine synthetase and ATP converted the 75Se-labeled amino acid to a radioactive basic product that was indistinguishable from authentic Se-adenosylselenomethionine by ion exchange and TLC . The native selenoenzyme, Mr 155,000-158,000, is composed of four subunits of Mr 38,000-40,000 . Thiolase of similar molecular weight that is less acidic and lacks selenium is also produced by C . kluyveri . The factors that control the relative levels of the two enzymes in the cell have not been identified. Arch Microbiol, 1982 Aug, 132(2), 155 - 8 Biosynthesis of vitamin B12 . Different pathways in some aerobic and anaerobic microorganisms; Hollriegl V et al.; Radioactivity from {1'-14C}riboflavin was incorporated into the 5,6-dimethylbenzimidazole moiety of Vitamin B12 in the aerobes Bacillus megaterium, Nocardia rugosa and Streptomyces sp . as well as in the aerotolerant anaerobe Propionibacterium freudenreichii, but not in the anaerobe Eubacterium limosum . As recently published for E . limosum, also in the anaerobe Clostridium barkeri radioactivity from {1-14C}glycine and {2-14C}glycine was found in the 5,6-dimethylbenzimidazole moiety, but not in the corrin moiety . The addition of L-{methyl-14C}methionine to C . barkeri led to the labeling of the corrin moiety and the 5,6-dimethylbenzimidazole moiety, showing that the seven "extra" methyl groups in the corrin ring as well as the two methyl groups of the base part originate from this precursor . In Clostridium thermoaceticum, forming the vitamin B12 analog 5-methoxybenzimidazolylcobamide, {1-14C}glycine and {2-14C}glycine were also incorporated into the 5-methoxybenzimidazole moiety, but not into the corrin ring . In E . limosum L-{U-14C}glutamate led to the labeling of the corrin ring of vitamin B12, but not of its base moiety . These results together with data from the literature indicate that a common biosynthetic pathway might exist for the corrinoid biosynthesis in aerobic microorganisms, and in those aerotolerant anaerobes like the Propionibacteria, which form the 5,6-dimethylbenzimidazole moiety of vitamin B12 only under aerobic conditions . They also show that this pathway differs from the pathway found in anaerobic bacteria. Appl Environ Microbiol, 1982 Aug, 44(2), 334 - 8 Metabiotic effect of Bacillus licheniformis on Clostridium botulinum: implications for home-canned tomatoes; Montville TJ; The metabiotic effect of Bacillus licheniformis on Clostridium botulinum was examined . B . licheniformis elevated the pH of a model system with an initial pH of 4.4 so that C . botulinum grew and produced toxin . Toxin production was observed when spores from both species were coinoculated at levels as low as 10 spores per ml . When pint jars of tomatoes were used, canner size contributed to a 10,000-fold difference in the lethality of a boiling water bath process on B . licheniformis spores . Botulinal toxin was not detected in pH-elevated jars of tomatoes containing C . botulinum spores. Appl Environ Microbiol, 1982 Aug, 44(2), 447 - 52 Histamine-producing bacteria in decomposing skipjack tuna (Katsuwonus pelamis); Yoshinaga DH et al.; Spoilage in skipjack tuna (Katsuwonus pelamis) was studied under controlled conditions by incubating whole, fresh fish in seawater at 38 degrees C, the optimum temperature for histamine formation . Bacterial isolates were obtained from the loin tissue of a decomposing tuna containing 134 mg of histamine per 100 g and a total anaerobic count of 3.5 x 10(5)/g after incubation for 24 h . Over 92% of the 134 isolates obtained were facultatively or obligately anaerobic bacteria . Eighteen isolates produced histamine in culture media containing histidine, and these were identified as Clostridium perfringens, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, and Vibrio alginolyticus . Histidine decarboxylase activity of several isolates was measured in a tuna broth medium and with resting cells suspended in a buffered histidine solution. Infect Immun, 1982 Aug, 37(2), 486 - 91 Quantitation of binding and subcellular distribution of Clostridium perfringens enterotoxin in rat liver cells; Tolleshaug H et al.; Binding of enterotoxin from Clostridium perfringens type A was studied in suspensions of parenchymal and nonparenchymal cells from rat liver . In hepatocytes, 1.5 X 10(6) specific binding sites per cell with an association constant of 3.2 X 10(6) M-1 were found . About 1% of the added toxin was nonspecifically bound to the hepatocytes . At concentrations of toxin below 0.1 micrograms/ml, 80% of the toxin density of 7 X 10(6) cells per ml . Binding did not increase after the cells became permeable to the toxin . Subcellular fractionation in a sucrose gradient produced no evidence for binding to parts of the cell other than the plasma membrane . The degree of binding to nonparenchymal cells was less than 10% of the binding to hepatocytes. Eur J Biochem, 1982 Aug, 126(1), 35 - 42 Purification of L-glutamate-dependent citrate lyase from Clostridium sphenoides and electron microscopic analysis of citrate lyase isolated from Rhodopseudomonas gelatinosa, Streptococcus diacetilactis and C . sphenoides; Antranikian G et al.; Citrate lyase from Clostridium sphenoides was purified 72-fold with a yield of 11% . In contrast to citrate lyase from other sources the activity of this enzyme was strictly dependent on the presence of L-glutamate . The purified enzyme was only stable in the presence of 150 mM L-glutamate or 7 mM L-glutamate plus glycerol, sucrose or bovine serum albumin . Changes of the L-glutamate pool and of enzyme activity in growing cells of C . sphenoides indicated that citrate lyase activity in this organism was regulated by the intracellular L-glutamate concentration . Citrate lyase isolated from C . sphenoides, Rhodopseudomonas gelatinosa and Streptococcus diacetilactis was investigated by electron microscopy using the negative staining technique . Three different projections of enzyme molecules were observed: 'star' form, 'ring' form and 'triangle' form . In samples from R . gelatinosa and S . diacetilactis, star and ring forms occurred in a ratio of about 1:9 . Using the enzyme from S . diacetilactis it was demonstrated that this ratio could be altered in favour of the star form by the addition of citrate or tricarballylate . The triangle form was observed in less than 1% of all evaluated molecules and may represent a transition form . In lyase samples from C . sphenoides there existed a correlation between enzyme activity and the proportion of stars and rings at varying concentrations of L-glutamate. Biochemistry, 1982 Jul 20, 21(15), 3530 - 7 Monovalent cation induced reassociation of formyltetrahydrofolate synthetase monitored by Rayleigh light scattering and enzymic activity; de Renobales M et al.; It has been previously established that formyltetrahydrofolate synthetase isolated from Clostridium cylindrosporum is reversibly dissociated and inactivated in the absence of certain monovalent cations . In the present paper, the reassociation of monomeric, inactive enzyme to form tetrameric, active enzyme was monitored by Rayleigh light scattering and enzymic activity . Light-scattering measurements confirmed that the active enzyme is composed of four subunits of equal weight . With the assumption that the results of analytical ultracentrifugation are correct--that monomers and tetramers are the only species ever present at appreciable levels--the amount of tetramer formed during reassociation was calculated from the light-scattering data . Evidence for the accumulation of catalytically active intermediates was obtained by comparing the rate of association of monomers (detected by light scattering) to the rate of return of enzymic activity . The accumulation of intermediates was most strikingly seen at low monovalent cation concentration at low ionic strength . Evidence is also presented that sedimentation favors reassociation of the enzyme . The reassociation data were fit to a second-order reversible rate equation . Interestingly, although the data were derived from the same experiments, the kinetic plot based on light-scattering measurements yielded a straight line function with an abrupt change in slope about 10 min after initiation of reassociation, while plots based on enzymic activity measurements gave a single slope. Pathology, 1982 Jul, 14(3), 317 - 22 Toxin-induced cell membrane injury in guinea pigs given lincomycin; Lee SP et al.; Guinea pigs treated with lincomycin developed colitis, acute cholecystitis and abnormalities in red blood cell morphology . The present study was designed to study the production of clostridial toxins after lincomycin treatment . Lincomycin produced abnormalities in conventional but not in germ-free guinea pigs . Clostridium difficile was cultured from cecal contents of conventional guinea pigs treated with lincomycin . Cecal filtrate from sick guinea pigs was subjected to Sepharose 4B-CL and Sephadex G-200 column chromatography, yielding a partially purified toxin . Both cecal filtrate and partially purified toxin samples contained a heat labile substance which was cytotoxic to human lung fibroblast Wl-38 cells, and which was neutralized by Clostridium sordelli antitoxin . Toxin samples given orally or intraperitoneally killed normal guinea pigs . Finally, toxin samples induced red cell membrane changes in vitro as well as producing features of acute inflammation in healthy explants of guinea pig cecum and gallbladder in organ culture . Lincomycin treated guinea pigs produced Clostridium difficile toxin(s) which in turn caused diffuse cell membrane injury. Biochimie, 1982 Jul, 64(7), 503 - 7 Non-heme iron proteins of Desulfovibrio: the primary structure of ferredoxin I from Desulfovibrio africanus; Bruschi M et al.; Three different ferredoxins have been isolated from the sulfate reducing bacterium, Desulfovibrio africanus . The present paper describes the complete amino acid sequence of D . africanus ferredoxin I . This sequence was determined using automatic protein sequencing in liquid phase and in solid phase . The 61 amino acid residues of the sequence have been aligned with the aid of peptides obtained by cyanogen bromide, cleavage and by tryptic hydrolysis . This ferredoxin which contains 4 cysteine residues represents the most simple case of one (4 Fe-4 S) cluster ferredoxin . A comparison of D . africanus ferredoxin I with D . gigas and Clostridium pasteurianum ferredoxins is presented in terms of structural and possible evolutionary relationships. J Gen Microbiol, 1982 Jul, 128(7), 1457 - 66 Selenium-dependent growth and glycine fermentation by Clostridium purinolyticum; Durre P et al.; Clostridium purinolyticum fermented glycine as a sole carbon and energy source according to the equation: 4 Glycine + 2H2O leads to 3 Acetate + 2CO2 + 4NH3 . The organism required adenine as a supplement and selenium compounds as micronutrients for growth . The molar growth yield on glycine was 6.5 g dry wt . Radiochemical and enzymic investigations revealed a new fermentation pathway for glycine in which 1 mol glycine was completely oxidized to CO2 and the generated reducing equivalents were used to reduce a further 3 mol glycine to acetate via the glycine reductase system . This reaction was associated with the formation of ATP. J Lipid Res, 1982 Jul, 23(5), 726 - 32 The metabolism of primary, 7-oxo, and 7 beta-hydroxy bile acids by Clostridium absonum; Sutherland JD et al.; Clostridium absonum was shown to metabolize primary bile acids to give rise to both 7-oxo bile acids and 7 beta-hydroxy (urso) bile acids . At relatively low redox potential (Eh) values, high yields of urso bile acids were achieved (60-75%) . If, however, the Eh value of the culture was allowed to rise above approximately -100 mv, the 7-oxo bile acid would tend to predominate (more than 75%) and the "death phase" was accelerated . Growth of C . absonum in sterile graduated cylinders instead of in conventional Erlenmeyer flasks was effective in delaying the rise in Eh value with time (which appears largely due to diffusion of atmospheric oxygen into the medium) and in preserving a higher viable count of organisms . It is proposed that the formation of excess amounts of 7-oxo bile acid is a manifestation of oxygen toxicity and that it could be mediated by an increasing intracellular NADP:NADPH ratio . Additionally, the reaction: primary bile acid in equilibrium oxo bile acid in equilibrium urso bile acid was shown to be partially reversible . When the organisms were grown with {24-(14)C}chenodeoxycholic, -cholic, or -7-keto-lithocholic acid, this reaction could be clearly demonstrated . The addition of an equimolar concentration of deoxycholic acid (which itself is not metabolized) effectively enhanced the rate of bioconversion of cholate and 7-keto-lithocholic, but not chenodeoxycholate (whose rate of bioconversion was the fastest of the three) . When the organisms were grown with urso bile acids (ursocholic or ursodeoxycholic) or with 7-keto-deoxycholic acid, very little metabolism occurred unless deoxycholic acid was added which induced formation of primary and keto bile acids . In all cases, formation of oxo bile acid from primary or urso bile acid occurred as the Eh value of the medium rose with time and could thus be delayed by the use of a cylinder instead of a flask for growing the culture . These results were rationalized by demonstrating that induction of 7 alpha- and 7 beta-hydroxysteroid dehydrogenase is strongly mediated by chenodeoxycholic and deoxycholic acids, weakly mediated by cholic and 7-keto-lithocholic acids, and ineffective with 7-keto-deoxycholic, ursocholic, and ursodeoxycholic acids. Can J Surg, 1982 Jul, 25(4), 438 - 42 Clostridium difficile: epidemiology and clinical features; Marrie TJ et al.; To determine the epidemiologic features of Clostridium difficile in Halifax, Nova Scotia, the authors studied two groups of hospitalized patients, one group of outpatients and a fourth group of 54 healthy subjects . The first group consisted of 29 patients with diarrhea, whose stool was found to contain C . difficile or its cytotoxin, or both . Twenty-two underwent sigmoidoscopic examinations; of these, 18 had abnormal colonic mucosa and 6 of the 18 had pseudomembranous colitis . In the second group of 127 patients on general medical wards, 22 (17%) carried C . difficile . Thirteen of the 22 had diarrhea, and 3 had pseudomembranous colitis . Clustering of patients with C . difficile was evident . In vitro production of toxin by isolates of C . difficile from these patients was more likely if antibiotics had been given . Only 1 (4.5%) of the 22 outpatients with various gastrointestinal disorders (group 3) and none of the 54 healthy subjects (group 4) carried C . difficile . The clinical spectrum of infection with C . difficile extended from asymptomatic patients to those with nonspecific colitis and pseudomembranous colitis. Am J Gastroenterol, 1982 Jul, 77(7), 491 - 3 Antibiotic-associated hemorrhagic colitis; Gould PC et al.; Pseudomembranous colitis arising from Clostridium difficile super-infection after treatment with various antibiotics is a well-defined portion of the pathological spectrum of antibiotic related bowel injury . We have observed two patients with hemorrhagic colitis associated with the use of penicillin derivatives . The colitis was characterized by predominant right-sided involvement, sparing of the rectum and distal colon, absence of pseudomembrane formation, and presence of marked hemorrhage in the lamina propria . Discontinuation of antibiotics resulted in prompt resolution . Early colonoscopic examination was essential in establishing the diagnosis . Follow-up examination at 9 days demonstrated complete histological and endoscopic resolution. Biokhimiia, 1982 Jul, 47(7), 1159 - 64 {Catabolism of threonine in the bacterium Clostridium sticklandii}; Golovchenko NP et al.; Catabolism of L-threonine in the anaerobic bacteria Cl . sticklandii has been studied . Degradation of this amino acid was shown to occur with participation of the following enzymes: NAD-dependent L-threonine-3-dehydrogenase (EC 1.1.1.103), glycine acetyltransferase (EC 2.3.1.29), phosphotransacetylase (EC 2.3.1.8) and acetate kinase (EC 2.7.2.1) . The presence of the first two enzymes in Clostridia has been shown for the first time . A scheme of threonine oxidation down to acetic acid and glycine by Cl . sticklandii was proposed . The oxidation of one threonine molecule is coupled with phosphorylation of one ADP molecule and reduction of one NAD+ molecule. J Bacteriol, 1982 Jul, 151(1), 507 - 9 Levels of enzymes involved in the synthesis of acetate from CO2 in Clostridium thermoautotrophicum; Clark JE et al.; The acetogenic bacterium Clostridium thermoautotrophicum, grown on methanol, glucose, or CO2-H2, contained high levels of corrinoids, formate dehydrogenase, tetrahydrofolate enzymes, carbon monoxide dehydrogenase, and hydrogenase . Cell-free extracts catalyzed pyruvate-dependent formation of acetate from methyltetrahydrofolate . These results suggest that C . thermoautotrophicum synthesizes acetate from CO2 via a formate-tetrahydrofolate-corrinoid pathway. Int Surg, 1982 Jul-Sep, 67(3), 271 - 3 Clostridium perfringens meningitis; Ho KL; In this report, primary Clostridium perfringens meningitis, a rare condition, is described in a 62-year-old man who developed fatal clostridial meningitis following surgical evacuation of a subdural hematoma . This is the third case report of clostridial meningitis as a complication of craniotomy . Clostridial infection should be considered in any patient with meningitis with a history of surgical intervention, or head injury, even if only slight. Can J Microbiol, 1982 Jul, 28(7), 860 - 73 Characterization of bacteriocin 28 produced by Clostridium perfringens; Li AW et al.; Bacteriocin 28, produced by Clostridium perfringens, was characterized by gel filtration and sodium dodecyl sulfate - polyacrylamide gel electrophoresis as a glycoprotein with a molecule weight of approximately 100,000 . Density gradient centrifugation suggested a lower weight of 84,000 . The bacteriocin bound firmly to phenyl-Sepharose CL-4B gel, indicating hydrophobic properties, and elution from this gel with ethylene glycol clearly separated bacteriocin from the alpha and theta toxins of C . perfringens, the latter of which was also hydrophobic . Bacteriocin 28 was immunogenic, inducing neutralizing and precipitating antibodies, and possessed three isoelectric points: 7.37, 7.05, and 5.4 . Amino acid and carbohydrate analysis of the active material showed a composition of 15 amino acids and several carbohydrates . The molecule demonstrated instability with increasing purification, and several approaches to purification are described. Infect Immun, 1982 Jul, 37(1), 336 - 43 Gram-positive bacteria-induced granulocytopenia and pulmonary leukostasis in rabbits; Goldblum SE et al.; Pneumococci have been shown to induce granulocytopenia and pulmonary leukostasis which might contribute to morbidity and mortality in pneumococcal sepsis . We studied whether other gram-positive species (groups A and B streptococci, Staphylococcus aureus, Bacillus cereus, and Clostridium perfringens) could also induce these phenomena . Rabbits were challenged with nonviable preparations of each species, and all five species induced profound granulocytopenia (mean decrease of 90%) and pulmonary leukostasis . In vitro studies of serum incubated with these species of bacteria showed a mean consumption of total hemolytic complement of 90%, a mean increase of chemotactic activity for granulocytes of 204%, and a mean augmentation of granulocyte adherence of 45% (compared with 18% for the control) . Infusion of sonicate-exposed sera induced granulocytopenia in recipient rabbits . Thus, several nonviable gram-positive species can interact with serum to activate the complement system, generate C5a bioactivity, augment granulocyte adherence, and generate a neutropenia-inducing factor . These alterations may contribute to granulocytopenia or pulmonary leukostasis, which may play a role in the morbidity and mortality associated with gram-positive bacterial infections. Can J Microbiol, 1982 Jul, 28(7), 851 - 9 Effect of purine derivatives, papaverine hydrochloride, and imidazole on enterotoxin formation by Clostridium perfringens type A; Craven SE et al.; The percentage sporulation and enterotoxin specific activity were improved for all of five Clostridium perfringens strains, and numbers of heat-resistant spores were improved for four of five strains by replacing proteose peptone with peptone in Duncan-Strong (DS) medium . When raffinose replaced starch in DS, peptone was superior to proteose peptone in increasing percentage sporulation, numbers of heat-resistant spores, and enterotoxin formation for four of five strains . Enterotoxin levels for a strain varied when different lots of the same peptone were used . Additional experiments were conducted with three C . perfringens strains grown in DS medium with peptone . Enterotoxin specific activity was increased for three strains by adding papaverine (hydrochloride crystalline), for two strains by adding each of caffeine and 3-isobutyl-l-methylxanthine, for one strain by adding each of theophylline, 6-mercaptopurine, and 2-amino-6-mercaptopurine, and for none of the strains by adding imidazole . When enterotoxin formation was improved for a strain by one of the compounds, percentage sporulation increased, but growth decreased . Effective compounds also increased numbers of heat-resistant spores for strains H6 and R42, but slightly or not at all for strain E13 . The action of these compounds was concentration dependent, with the optimal concentration differing between compounds and between strains grown in the presence of the same compound. Biochim Biophys Acta, 1982 Jun 14, 688(2), 453 - 9 Reactivity of glycoconjugates in membranes . I . Determination of transbilayer distribution of gangliosides in lipid vesicles by chemical methods; Thomas PD et al.; Two simple chemical methods are described for the determination of the transbilayer distribution of gangliosides GD1a and GM1 in phosphatidylcholine vesicles . The data presented here show an increase in the percentage of GD1a exposed on the outer surface of vesicles with increasing mole fraction of GD1a . The percentage of GD1a exposed on 1:50 and 1:5 GD1a-dipalmitoylphosphatidylcholine vesicles were found to be 67% and 83%, respectively . The same trend is seen for vesicles with dimyristoylphosphatidylcholine and distearoylphosphatidylcholine . Extrapolation of the data to infinite dilution gives 65% of GD1a exposed on the surface of GD1a-dipalmitoylphosphatidylcholine vesicles . The results indicate that composition-dependent changes in transbilayer distribution of GD1a can only partly account for the observed increase in the reactivity of GD1a in vesicles towards neuraminidase from clostridium perfringens as the ratio of GD1a to phosphatidylcholine increases. J Gen Microbiol, 1982 Jun, 128(Pt 6), 1365 - 70 Immunochemistry of the cell-surface carbohydrate antigens of Clostridium difficile; Poxton IR et al.; Two carbohydrate cell-surface antigens were extracted from Clostridium difficile . One was extracted from pure cell walls by NaOH and contained glucose, mannose, galactosamine and phosphate in the approximate molar proportions of 2:0.65:1:0.63 . The other antigen was extracted with phenol from the disrupted contents of whole cells and purified by chromatography on Sepharose 6B and an immunoabsorbent column; it contained glucose, glucosamine, phosphate and fatty acid in the approximate molar proportio